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název v anglickém jazyce není uveden
Krásná, Luboslava ; Veselý, Pavel (advisor) ; Křepela, Evžen (referee) ; Červinka, Miroslav (referee)
An in vitro cultivation technique has been developed that allows to reproducibly obtain and propagate a heterogenous population of epithelial cells from samples of normal mammary glands, breast tumours and subcutaneous metastases of breast cancer. The epithelial origin of cultured cells was proved by their cytokeratin profile, and EMA and ESA protein expression. The technique uses a feeder layer of irradiated NIH 3T3 cells to support clonal growth of even single isolated cells. Cultures were successfully established from 96% of surgical tumour biopsy specimens (69 out of 72, volume of sample about 1 cm3) and 67% of true-cut biopsy specimens (28 out of 42, volume 0.01 cm3). Two and more in vitro passages were obtained with 74% (53 out of 72) of surgical specimens and 31% (13 out of 42) of truecut biopsy specimens. This is the first report of successful in vitro propagation of cells obtained from breast cancer via true-cut biopsy. In cell populations analyzed for the growth properties, the culture lifetime, related to the number of colony-forming cells, varied for cells from benign tumours between 22 and 40 cell populations and for malignant tumours between 21 and 51. The population doubling time varied betwen 18 and 62 hours for benign cultures and 10 and 127 hours for malignant cultures, with average 32...
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Preparation of contructs for transgenic expression of DPP-IV and FAP
Košek, Dalibor ; Bezouška, Karel (advisor) ; Křepela, Evžen (referee)
Preparation of contructs for transgenic expression of DPP-IV and FAP Bc. Dalibor Košek Abstract: DASH (Dipeptidyl peptidase-IV Activity and/or Structure Homologues) protein group involves multi-funcional molecules typically bearing enzymatic activity similar to the Dipeptidyl peptidase-IV (DPP-IV, EC 3.4.14.5, identical with lymphocyte differentiation antigen CD26). In general, they cleave multiple regulatory as well as structural peptides and proteins, possessing proline residue on the penultimate position from the N-terminus. We focused on two members of this group: canonical DPP-IV and Fibroblast activation protein alpha (FAP-α). Both are typically type II plasma membrane proteins with specific tissue distribution. Soluble extrecellular forms have also been identified. Available knowledge suggest important roles of these proteins in oncogenesis, executed by their enzymatic activity but also by non-proteolytic interactions. To study their role in gliomagenesis we designed several experimental models exploiting astrocytoma cell lines with defined DPP-IV or FAP-α phenotype. Enzymatically inactive forms and analogues with different subcellular distribution will also be included. These models will allow to assess the impact of DPP-IV and FAP-α on the glial tumor development and the importance of their...
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The application of electrophoretic and chromatographic methods in clinical research
Fauknerová Matějčková, Jana ; Samcová, Eva (advisor) ; Pacáková, Věra (referee) ; Křepela, Evžen (referee)
- The application of electrophoretic and chromatographic methods in clinical research RNDr. Jana Fauknerová Matějčková Malondialdehyde (MDA) is considered to be the main biomarker for oxidative damage to biomembranes and an elevated level of this compound can act as an indicator for a number of diseases. I determined MDA using a HPLC method based on derivatization of blood plasma samples with 2,4-dinitrophenylhydrazine. I used HPLC to separate the MDA derivatives from the other components of the blood plasma and detected them at 307 nm. The MDA analysis time did not exceed 4.5 min. The sensitivity (detection limit 0.27 mol l-1 ) and repeatability of the determination of MDA are sufficient for monitoring the MDA level in real blood plasma samples. My work also describes monitoring the level of the low-molecular secondary antioxidant of uric acid, whose concentration in the blood attains values of 140 to 350 µmol l-1 and the secondary enzymatic antioxidant of superoxide dismutase (SOD). The uric acid level was analysed by the capillary electrophoresis and spectrophotometric methods, where the two methods yielded comparable results. SOD was analysed using a spectrophotometric kit. The MDA levels were measured for vegans (who refuse to eat any food of animal origin, meat, eggs and also dairy...
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Expression and function of serpin B9 in lung carcer cells
Roušalová, Ilona ; Křepela, Evžen (advisor) ; Šedo, Aleksi (referee) ; Kotyza, Jaromír (referee)
Background: Granzyme B (GrB) is a key proapoptotic secretory protease of CTLs and NK cells. Its specific proapoptotic effects in cancer cells can be blocked by increased expression of serpinB9. SerpinB9 gene expression can be transcriptionally upregulated by some interleukins and by the oestrogen activated oestrogen receptor-α (ERα) in cells which express ERα protein. The aims of my thesis were to evaluate the expression of SB9 and to examine its inhibitory activity against exogenous active GrB in non-small cell lung carcinoma (NSCLC) cell lines and tissues. To analyse the expression status of GrB mRNA in NSCLC cell lines and tissues. To investigate the role of estradiol-17β (E2), selected ILs and DNA methylation in regulation of SB9 expression in NSCLC cells. The apoptosome apparatus is a cell death signalling platform activates the initiator procaspase-9. Activation of the apoptosome apparatus is often impaired in various types of cancer but the molecular basis of its suppression is still unknown. APIP and UACA/nucling belong to the endogenous regulators of apoptosome apparatus. The aim of my thesis was to investigate whether DNA methylation is involved in the transcriptional regulation of expression of APIP and UACA genes in NSCLC cell lines. Methods: Following methods were used in this thesis:...
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Expression of apoptosome pathway regulators and activation of apoptosome apparatus in non-small cell lung carcinoma
Moravčíková, Erika ; Křepela, Evžen (advisor) ; Kovář, Jan (referee) ; Kotyza, Jaromír (referee)
Background: The apoptosome pathway is interesting as potential therapeutic target because it plays an important role in the cancer chemotherapy- and biological therapy-induced apoptosis as well as in amplifying the death receptor and cytotoxic-granule-induced pathways. The functionality of apoptosome apparatus in non-small cell lung carcinoma (NSCLC) cells and tissues is often impaired due to defects in apoptosome pathway by changes in expression and/or acquired mutations and/or modifications of apoptosome components or its regulators that play a significant role in cancer cell proliferation and treatment unresponsiveness. Therefore, this thesis is aimed at the investigation of the expression status of apoptosome pathway regulators (survivin, HBXIP, XIAP, APIP, and UACA) and of the readiness of apoptosome apparatus activation in non-small cell lung carcinoma cells and tissues. Methods: Following methods were used in this thesis: isolation and quantification of total RNA, real-time RT-PCR analysis, preparation of cell-free cytosol samples and extracts from cells and tissues, gel-filtration chromatography, Western blot analysis, enzyme analyses and cell culture techniques. Results and conclusion: Non-small cell lung carcinoma has a higher predisposition to apoptosome-mediated apoptosis than normal...
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The application of electrophoretic and chromatographic methods in clinical research
Fauknerová Matějčková, Jana ; Samcová, Eva (advisor) ; Pacáková, Věra (referee) ; Křepela, Evžen (referee)
- The application of electrophoretic and chromatographic methods in clinical research RNDr. Jana Fauknerová Matějčková Malondialdehyde (MDA) is considered to be the main biomarker for oxidative damage to biomembranes and an elevated level of this compound can act as an indicator for a number of diseases. I determined MDA using a HPLC method based on derivatization of blood plasma samples with 2,4-dinitrophenylhydrazine. I used HPLC to separate the MDA derivatives from the other components of the blood plasma and detected them at 307 nm. The MDA analysis time did not exceed 4.5 min. The sensitivity (detection limit 0.27 mol l-1 ) and repeatability of the determination of MDA are sufficient for monitoring the MDA level in real blood plasma samples. My work also describes monitoring the level of the low-molecular secondary antioxidant of uric acid, whose concentration in the blood attains values of 140 to 350 µmol l-1 and the secondary enzymatic antioxidant of superoxide dismutase (SOD). The uric acid level was analysed by the capillary electrophoresis and spectrophotometric methods, where the two methods yielded comparable results. SOD was analysed using a spectrophotometric kit. The MDA levels were measured for vegans (who refuse to eat any food of animal origin, meat, eggs and also dairy...
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Expression of apoptosome pathway regulators and activation of apoptosome apparatus in non-small cell lung carcinoma
Moravčíková, Erika ; Křepela, Evžen (advisor) ; Kovář, Jan (referee) ; Kotyza, Jaromír (referee)
Background: The apoptosome pathway is interesting as potential therapeutic target because it plays an important role in the cancer chemotherapy- and biological therapy-induced apoptosis as well as in amplifying the death receptor and cytotoxic-granule-induced pathways. The functionality of apoptosome apparatus in non-small cell lung carcinoma (NSCLC) cells and tissues is often impaired due to defects in apoptosome pathway by changes in expression and/or acquired mutations and/or modifications of apoptosome components or its regulators that play a significant role in cancer cell proliferation and treatment unresponsiveness. Therefore, this thesis is aimed at the investigation of the expression status of apoptosome pathway regulators (survivin, HBXIP, XIAP, APIP, and UACA) and of the readiness of apoptosome apparatus activation in non-small cell lung carcinoma cells and tissues. Methods: Following methods were used in this thesis: isolation and quantification of total RNA, real-time RT-PCR analysis, preparation of cell-free cytosol samples and extracts from cells and tissues, gel-filtration chromatography, Western blot analysis, enzyme analyses and cell culture techniques. Results and conclusion: Non-small cell lung carcinoma has a higher predisposition to apoptosome-mediated apoptosis than normal...
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Expression and function of serpin B9 in lung carcer cells
Roušalová, Ilona ; Křepela, Evžen (advisor) ; Šedo, Aleksi (referee) ; Kotyza, Jaromír (referee)
Background: Granzyme B (GrB) is a key proapoptotic secretory protease of CTLs and NK cells. Its specific proapoptotic effects in cancer cells can be blocked by increased expression of serpinB9. SerpinB9 gene expression can be transcriptionally upregulated by some interleukins and by the oestrogen activated oestrogen receptor-α (ERα) in cells which express ERα protein. The aims of my thesis were to evaluate the expression of SB9 and to examine its inhibitory activity against exogenous active GrB in non-small cell lung carcinoma (NSCLC) cell lines and tissues. To analyse the expression status of GrB mRNA in NSCLC cell lines and tissues. To investigate the role of estradiol-17β (E2), selected ILs and DNA methylation in regulation of SB9 expression in NSCLC cells. The apoptosome apparatus is a cell death signalling platform activates the initiator procaspase-9. Activation of the apoptosome apparatus is often impaired in various types of cancer but the molecular basis of its suppression is still unknown. APIP and UACA/nucling belong to the endogenous regulators of apoptosome apparatus. The aim of my thesis was to investigate whether DNA methylation is involved in the transcriptional regulation of expression of APIP and UACA genes in NSCLC cell lines. Methods: Following methods were used in this thesis:...
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Preparation of contructs for transgenic expression of DPP-IV and FAP
Košek, Dalibor ; Bezouška, Karel (advisor) ; Křepela, Evžen (referee)
Preparation of contructs for transgenic expression of DPP-IV and FAP Bc. Dalibor Košek Abstract: DASH (Dipeptidyl peptidase-IV Activity and/or Structure Homologues) protein group involves multi-funcional molecules typically bearing enzymatic activity similar to the Dipeptidyl peptidase-IV (DPP-IV, EC 3.4.14.5, identical with lymphocyte differentiation antigen CD26). In general, they cleave multiple regulatory as well as structural peptides and proteins, possessing proline residue on the penultimate position from the N-terminus. We focused on two members of this group: canonical DPP-IV and Fibroblast activation protein alpha (FAP-α). Both are typically type II plasma membrane proteins with specific tissue distribution. Soluble extrecellular forms have also been identified. Available knowledge suggest important roles of these proteins in oncogenesis, executed by their enzymatic activity but also by non-proteolytic interactions. To study their role in gliomagenesis we designed several experimental models exploiting astrocytoma cell lines with defined DPP-IV or FAP-α phenotype. Enzymatically inactive forms and analogues with different subcellular distribution will also be included. These models will allow to assess the impact of DPP-IV and FAP-α on the glial tumor development and the importance of their...
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název v anglickém jazyce není uveden
Krásná, Luboslava ; Veselý, Pavel (advisor) ; Křepela, Evžen (referee) ; Červinka, Miroslav (referee)
An in vitro cultivation technique has been developed that allows to reproducibly obtain and propagate a heterogenous population of epithelial cells from samples of normal mammary glands, breast tumours and subcutaneous metastases of breast cancer. The epithelial origin of cultured cells was proved by their cytokeratin profile, and EMA and ESA protein expression. The technique uses a feeder layer of irradiated NIH 3T3 cells to support clonal growth of even single isolated cells. Cultures were successfully established from 96% of surgical tumour biopsy specimens (69 out of 72, volume of sample about 1 cm3) and 67% of true-cut biopsy specimens (28 out of 42, volume 0.01 cm3). Two and more in vitro passages were obtained with 74% (53 out of 72) of surgical specimens and 31% (13 out of 42) of truecut biopsy specimens. This is the first report of successful in vitro propagation of cells obtained from breast cancer via true-cut biopsy. In cell populations analyzed for the growth properties, the culture lifetime, related to the number of colony-forming cells, varied for cells from benign tumours between 22 and 40 cell populations and for malignant tumours between 21 and 51. The population doubling time varied betwen 18 and 62 hours for benign cultures and 10 and 127 hours for malignant cultures, with average 32...
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